This invention relates to proteins that bind nucleotide mismatches, DNAs encoding such proteins, and uses thereof, particularly in mismatch detection assays.
Mutation detection techniques provide powerful tools for the prediction and diagnosis of disease arising from one or more changes in a nucleotide sequence. Such changes generally occur in coding regions and result in protein products that are either inactive or are altered in their level or type of activity. Less commonly, mutations resulting in disease states occur in nucleotide sequences that do not encode a protein product; for example, mutations in repetitive DNA have been shown to be associated with such diseases as human fragile-X syndrome, spinal and bulbar muscular dystrophy, and myotonic dystrophy.
To detect mismatches at the nucleic acid level, a number of screens have been developed, many employing a technique commonly referred to as heteroduplex analysis. This technique involves the formation of a duplex between one strand of a control nucleic acid (typically, of wild-type sequence) and one strand of a test nucleic acid (for example, one suspected of including a mutation). The presence of a mismatch in the duplex is then revealed by any of a number of standard approaches, including RNAse A digestion, chemical cleavage, or PCR-based or primer extension-based techniques (reviewed in Cotton, Curr. Opinion in Biotech. 3: 24, 1992).
Another means by which mismatches may be identified in a heteroduplex molecule is by the use of mismatch-specific proteins. These proteins are capable of recognizing and either binding or cleaving at or near a site of mismatch. One particular class of mismatch-specific binding proteins is the "Mut" series of bacterial polypeptides. An exemplary member of this class, the MutS protein, has been reported to bind heteroduplex DNA at sites of certain base pair mismatches and at loops resulting from deletions or insertions of up to four bases in length (Parker et al., Proc. Natl. Acad. Sci. USA 89: 1730-1734, 1992; Modrich et al., U.S. Pat. No. 5,459,039, 1995).
Given the role played by mismatch-specific proteins in mutation detection assays, the isolation and characterization of additional proteins having the capacity to identify mutant sites in nucleic acid substrates is of great value. These proteins find use in assays for the diagnosis and prognosis of diseases such as cancer, and are also useful for perinatal screening for inherited diseases, differential diagnosis of diseases not readily detectable by conventional tests (for example, Marfan's syndrome), and the detection of genetic alleles (for example, for genetic mapping, tissue matching, or identification purposes).